DESCRIPTION (Verbatim from Applicant's Abstract): Our overall objective is to define the mechanism of action of cholera toxin (CT). While CT directly upregulates adenylate cyclase activity by catalyzing the ADP-ribosylation of Gs-alpha, thereby increasing 3',5'-adenosine monophosphate (cAMP) levels, equally important are stimulatory effects on arachidonic acid (AA) metabolism leading to increased production of eicosanoids (e.g., prostaglandin E2 [PGE2]). PGE2 stimulates adenylate cyclase, intestinal ion transport, and synthesis of cytokines (e.g., IL-6). Importantly, our recent results showed that CT and the CT B-subunit (CT-B) could stimulate AA metabolism independent of ADP-ribosylation of GS-alpha by signaling the expression of the plaa gene, which encodes phospholipase A2-activating protein (PLAA). After cloning the human plaa cDNA, we hyperexpressed the gene in prokaryotic and eukaryotic systems. Our experiments with plaa antisense oligonucleotides have shown that PLAA is an important participant in the mechanisms by which CT upregulates PLA2. Another novel finding includes the chemical reaction that occurs between PGE2 and imidazole derivatives (e.g., L-histidine). The formation of PGE2-imidazole (-histidine) covalent adducts was confirmed by NMR and mass spectrometry. We demonstrated that these adducts reduced cAMP levels in CT-treated CHO cells and reduced CT-induced fluid loss in a murine intestinal loop model. In this proposal, we will examine the role of PLAA, PGE2, and LTC4 in enhancing the stimulatory effect of cAMP on the secretory response in the small intestine. Intestinal cells (e.g., Paneth cells) forming PLAA in response to CT will be identified using in situ hybridization and immunohistochemistry. Further, the role of PLAA and Paneth cells will be established by using knockout mice. We also propose to define the regulatory mechanisms by which CT signals the rapid synthesis of PLAA mRNA. Using recombinant PLAA and expression of plaa cDNA in eukaryotic cells, we will evaluate its stimulatory effect on PLA2 activity. Increasing knowledge of the molecular events in cholera should lead to future strategies to control the hypersecretion of water and electrolytes.